Article date: 1985/10/15
PubMed ID: 2413862
Journal name: Biochemical pharmacology (ISSN: 0006-2952)
[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a bicyclic cage convulsant, binds to the anion gating mechanism of the GABA/benzodiazepine receptor chloride channel complex. Using a carefully calibrated radiation inactivation technique, the molecular weight of [35S]TBPS binding complexes from frozen rat cerebral cortex was estimated to be 137,000 daltons. The GABA agonist muscimol reduced [35S]TBPS binding to 0-10% of the control value, in a way which is independent of the radiation dose. This shows that the GABA receptor (Mw = 55,000 daltons) is included in the 137,000-dalton [35S]-TBPS binding complex; the [35S]TBPS binding protein alone accounts for 137,000-55,000 = 82,000 daltons. The pyrazolopyridazine etazolate (SQ 20.009) and etomidate in appropriate concentrations both reduced specific binding of [35S]TBPS. The ability of SQ 20.009 and etomidate to reduce [35S]TBPS binding was greatly reduced by exposure to low radiation doses, suggesting that SQ 20.009 and etomidate reduce [35S]TBPS binding by an allosteric mechanism requiring a molecular structure of 450,000-500,000 daltons. Benzodiazepine agonists (ethyl 4-methoxymethyl-6-benzyloxy-beta-carboline-3-carboxylate, ZK 93423) and inverse agonists (methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, DMCM) enhance and reduce [35S]TBPS binding, respectively, in repeatedly frozen and washed membrane preparations. The effects of ZK 93423 and DMCM on [35S]TBPS binding disappeared upon exposure of membranes to low radiation doses. This suggests that the benzodiazepine receptor site interacts allosterically with the [35S]TBPS binding site, requiring a molecular complex of at least c. 400,000 daltons. The [35S]TBPS site alone in these latter conditions of membrane preparation (repeatedly frozen/washed) revealed a molecular weight of 221,000 daltons (TBPS-site + GABA receptor + unknown structures). The number of binding sites for [35S]TBPS (145 pmol/g tissue) was only slightly higher than for [3H]flunitrazepam (130 pmol/g tissue) in cerebral cortex. These results are all consonant with the conclusion that the GABA/BZ receptor chloride channel complex is composed of highly integrated multimeric subunits, tentatively accounted for by a tetramic complex of molecular weight 548,000 daltons.
Author List: Nielsen M, Honore T, Braestrup C
Publication Types: Journal Article; Research Support, Non-U.S. Gov't
Substances mentioned in the article: Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Bridged-Ring Compounds; Carbolines; Chlorides; Ion Channels; Organophosphorus Compounds; Receptors, GABA-A; Sulfur Radioisotopes; Muscimol; 4-isopropylbicyclophosphate; tert-butylbicyclophosphorothionate; ZK 93423; Etazolate; Etomidate;
Mesh terms: Animals; Binding Sites/drug effects; Bridged Bicyclo Compounds/metabolism; Bridged Bicyclo Compounds, Heterocyclic; Bridged-Ring Compounds/metabolism; Carbolines/pharmacology; Cerebellum/drug effects; Cerebral Cortex/drug effects; Chlorides/metabolism; Etazolate/metabolism; Etomidate/metabolism; In Vitro Techniques; Ion Channels; Molecular Weight; Muscimol/metabolism; Organophosphorus Compounds/metabolism; Radioligand Assay; Rats; Receptors, GABA-A/drug effects; Sulfur Radioisotopes;